Project Summary/Abstract: Protein methylation is an epigenetic event that is regulated by >60 human protein methyltransferases (PMTs). Because many PMT-associated methylation events are invisible for conventional methods under designated cellular settings, our understanding of epigenetic roles of PMTs is very limited. Selective perturbation of PMTs is of critical need towards elucidating disease-specific roles of PMTs and formulating new therapy strategies. In the context of these technology and knowledge gaps, in the past five years, our laboratory has focused on developing novel technologies and implementing them to accurately annotate nonhistone methylation events and downstream molecular mechanisms of PMTs. Meanwhile, our laboratory has also devoted significant efforts to examine atomistic mechanisms underlying PMT-involved catalysis and leverage them to rationally develop PMT inhibitors via distinct modes of interaction. Here we plan to expand these findings. We envision characterizing nonhistone methylation events associated with transcription activation with integrated approaches including chemical labeling and Crisper/Cas9 editing. We will develop high-quality PMT inhibitors via SAM-competitive or covalent modes, in particular with consideration of dynamic conformational landscapes of PMTs. The completion of this proposal will unambiguously reveal molecular mechanisms of multiple PMTs in relevant cellular contexts and deliver high-quality PMT inhibitors for perturbation. The additional impact of our function-annotating and inhibitor-identifying strategies stems from their general applicability to other methyltransferases.